PROJECT SUMMARY Mechanical interactions between cells and extracellular matrix (ECM) drive fundamental processes such as morphogenesis, wound healing, and organization of bioengineered tissues. Our research focuses on how these interactions regulate corneal keratocyte behavior, through development of culture models that mimic the 3-D tissue environment, and use of multi-dimensional imaging approaches in vitro, in situ and in vivo. Research in the prior period using 3-D culture models demonstrated that matrix composition, stiffness and structure can influence corneal keratocyte mechanical behavior and patterning in response to wound healing cytokines and changes in Rho/Rac activation. In addition, using our custom-modified in vivo HRT-RCM confocal microscope combined with ex vivo fluorescence and second harmonic generation (SHG) imaging, we demonstrated for the first time that following freeze injury (FI) or lamellar keratectomy (LK) in the rabbit, migrating fibroblasts within the wounded stroma form long interconnected streams that often run in parallel, and that alignment of these cell streams is highly correlated with that of the collagen lamellae. In contrast, cells migrating on top of the stroma following LK form a randomly arranged, interconnected, meshwork. The biochemical factors which induce myofibroblast transformation and fibrotic tissue generation on top of the stroma following injury or refractive surgery have been studied extensively. However, little is known about biochemical and biophysical signals that regulate intra-stromal keratocyte behavior. The lamellar structure of the cornea, combined with powerful in vivo and ex vivo imaging capabilities, provides us with a unique opportunity to assess biophysical factors that regulate cell differentiation, migration and patterning within this tissue. Aim 1 will use in vivo confocal microscopy and in situ fluorescent/SHG imaging in the rabbit to: a) perform the first comprehensive comparison of intra-stromal and extra-stromal cell differentiation and patterning following photorefractive keratectomy (PRK), and b) investigate whether intra-stromal and extra- stromal phenotypes are differentially regulated. Aim 2 will investigate whether changes in ECM structure and stiffness modulate cell patterning and mechanical phenotype during stromal repopulation by comparing migration mechanisms in two distinct in vivo injury models. ECM structure and mechanical properties have become increasingly recognized as key factors in determining cell growth, differentiation and activity in a variety of cell types; thus our findings should have broad scientific impact. In order to isolate the specific factors regulating these in vivo processes, Aim 3 will assess how cytokines and downstream Rho/Rac signaling impact corneal keratocyte patterning, mechanical differentiation, fibronectin deposition and ECM reorganization using multiple novel experimental models in vitro. With this approach we hope to identify the key biochemical and biophysical signaling pathways that differentiate disruptive and non-disruptive cell patterning behavior within 3-D matrices, which may lead to new strategies to modulate cell behavior in vivo.